Propagation Methods
Protocorms and young seedlings of three Cypripedium
species. From left to right: C. reginae, C. guttatum, and
C.
formosanum. In each case, the culture tube occupying the width of the
photo is 2.5 cm (1 inch) across.
Approach
We propagate Cyp species using the basic methods of plant tissue culture
with one exception: Instead of beginning with explants, we start with seeds.
We are essentially beginning with the undifferentiated tissue of the seed
embryo, thereby avoiding the time-consuming step of slicing plant tissue
into small fragments.
Nutrition
Our basic method is the same as that used for the propagation of tropical
orchids from seed. In nature orchid seeds, which have essentially no stored
food reserves, obtain nutrients and energy by absorption from a symbiotic
fungus. Fungal filaments or hyphae invade orchid cells, only to be digested
by the cells. The term "symbiotic" is usually used for the relationship
between fungus and orchid; however, biologists have yet to find any benefit
to the fungus in the relationship. In in vitro culture, the fungus
is generally omitted, and the orchid seeds and plantlets obtain major and
minor nutrient elements from reagents added to an agar medium. Energy is
supplied by adding sugar, usually glucose, to the medium, and plant growth
regulators ("plant hormones") such as cytokinins or auxins are used as
necessary to stimulate germination and growth. In all in vitro orchid
culture, the medium and seed surface must be sterilized and sowing and
other manipulations carried out under aseptic conditions; otherwise the
cultures would quickly be overrun by competing organisms such as fungi
and bacteria.
Dormancy
One problem faced in the propagation and growth of Cyps not encountered
with most tropical orchids is dormancy. Cypripedium plants come
from temperate climates and exhibit winter dormancy. The seeds have a similar
dormancy that must be overcome before the seeds will germinate. Several
methods can be used to break dormancy including exposing the seeds to a
long cold period ("stratification"), stimulating the seeds with cytokinins,
and bleaching the seeds to remove germination-inhibiting substances. The
particular treatment varies considerably from one Cyp species to another,
and much research is still being done to find satisfactory methods for
certain species. Another approach to obtaining young plants is to remove
immature seeds whose embryos are still growing from the seed capsule of
the parent plant and place the seeds on the nutrient agar. In this way,
the embryos continue to grow in vitro without ever going through
a dormant state imposed by the parent plant.
Reflasking
As the young plants grow, they eventually reach a stage of development
at which they need to be transferred to fresh growth media. This reflasking
operation is necessary for several reasons: The plantlets may have different
needs for nutrients and growth regulators than they did at germination,
the young plants may have depleted nutrients in the medium or built up
waste products in the medium, or the seedlings may simply be too crowded.
The reflasking must be carried out under sterile conditions.
Removal from the flask and vernalization
Eventually, the seedlings reach a stage at which they cease growing. The
tips of the roots, normally bright yellow during active growth, turn brown.
At this stage, if the seedlings of most species are not removed from the
flask, they begin to die. Seedlings at this stage are dormant and must
be subjected to a lengthy period of low temperature to prepare them to
produce leaves. The photos below show seedlings in flasks at the stage
of maximum development. These seedlings are ready for removal from the
flasks and subsequent refrigeration.
The process of refrigerating seedlings at near-freezing temperatures
to overcome epicotyl dormancy is known as "vernalization." It is an essential
step in preparing the seedlings to produce leaves. The plants will not
make leaves and will eventually die if not given several months at low
temperature. We normally vernalize our seedlings after removing them from
the flasks by sealing the small plants in plastic freezer bags or in boxes
with a little water to prevent desiccation, and placing these containers
in a refrigerator. Careful monitoring of the refrigerator temperature is
important to prevent freezing the seedlings or premature leafing-out in
the refrigerator.
Seedlings at the proper growth stage for removal from
the flask. Left: C. californicum. Right: C. macranthos. The
flasks are 10 cm (4.0 inches) in diameter.
Future research
Cypripedium propagation is in its infancy. There is clearly much
more to learn about propagation methods. Seeds of some species germinate
reliably year after year. For other species, germination is unreliable
and seems to vary from year to year, from population to population for
a given species, and even from one clone to another within a population.
Seeds of some species have so far not been germinated in significant numbers.
Spangle Creek Labs is one of a number of organizations and individuals
in the world carrying on research aimed at improving germination and cultural
methods for these beautiful and fascinating plants. The number of artificially-propagated
Cyp plants and species should both increase substantially over the next
few years.
References
For people unfamiliar with the techniques of orchid micropropagation, P.A.
Thompson's primer Orchids from Seed (1996) is an excellent introduction.
This slim paperback was first published in 1977 by the Royal Botanic Gardens,
Kew and is available from the Shop at the American
Orchid Society.
If not daunted by Orchids from Seed, the beginning propagator
can find additional helpful information in the more advanced Asymbiotic
Technique of Orchid Seed Germination (1999). This book is available
from author Aaron Hicks at his Orchid
Seedbank Project site, where contents can be seen online.
Literature on Cyp propagation is still scanty. The following sources
should be helpful to people interested in attempting their own experiments
in Cyp propagation:
Malmgren, S. (1993). Orchids from Seed? Wildflower, 9(3),
32-34.
Rasmussen, H.N. (1995). Terrestrial orchids from seed to mycotrophic
plant. Cambridge: Cambridge University Press.
Sawyers, C.E. [ed.] (1990). North American native terrestrial orchid
propagation and production. Chadds Ford, Pennsylvania: Brandywine Conservancy.
Steele, W.K. (1995). Growing Cypripedium reginae from seed. American
Orchid Society Bulletin, 64, 382-91.
One of the best sources of information about propagating native orchids
is the proceedings volume of a 1996 conference on propagation of North
American terrestrial orchids that was held in Washington, DC. Many propagators
from North America and even Europe presented their methods for propagating
North American orchids including Cyps. This book, formerly published
by North American Native Orchid Conference, 14320 Poplar Hill Road, Germantown,
MD 20874, is now out of print, but is so useful that an effort to
locate a used copy may be worthwhile.
Finally, one of the best places to find sources of recent orchid propagation
information is the Internet. A little searching will turn up not
only Web sites with information but also the names of propagators that
are eager to share helpful tips.
Home